We began our work to discover genes required for expression or function of fusion oncogenes by using the results of a genome-wide RNAi screen designed to identify genes needed for the activity of the fusion oncoprotein EWS-FLI1. The development of this RNAi screen was a collaborative effort involving my laboratory and researchers from CCR's Pediatric Oncology Branch and the trans-NIH RNAi screening facility at the National Center for Advancing Translational Sciences (NCATS). EWS-FLI1 is an oncogenic transcription factor that occurs as a consequence of chromosomal rearrangements involving chromosomes 11 and 22. The majority of Ewing sarcomas (ES), cancers of the bone and soft tissue result from this chromosomal rearrangement. The genome-wide RNAi screen revealed the expression of the EWS-FLI1 fusion transcript is sensitive to the inhibition of the activity of specific splicing factors. We identified that expression of the EWS-FLI1 transcripts expressed in ES cells harboring translocations where the breakpoint in chromosome 22 occurs within a specific region of DNA requires the splicing factor HNRNPH1 to generate an in-frame mRNA. Depleting HNRNPH1 in ES cells where the breakpoint occurs within a specific region disrupts the expression and the activity of the EWS-FLI1 protein and reduces cell survival. Furthermore, we identified SF3B1, a major component of a protein complex required for splicing as a protein that ES cells are particularly dependent upon for cell survival. Ongoing studies are investigating the how HNRNPH1 facilitates the splicing of EWS-FLI1 in those ES cells that dependent upon its function with the long-term aim of identifying means in inhibiting this process. We are also investigating the function other proteins identified as required for the expression of the EWS-FLI1 protein, including one that regulates oncoprotein stability.